This invention relates to a purified enzyme and to a purification process for preparing the enzyme. In particular, it relates to the purified enzyme, deacetoxycephalosporin C synthetase (expandase) and to a process for preparing the enzyme in a high state of purity.
During the biosynthesis of cephalosporin C, deacetoxycephalosporin C (DAOC) synthetase mediates ring expansion of penicillin N to DAOC. The latter is then converted to deacetylcephalosporin C (DAC) by DAOC hydroxylase.
Partial purifications of the expandase enzyme from Cephalosporium acremonium, and Streptomyces clavuligerus have been described. Kupka, J., et al., FEMS Microbiol. Lett. 16, 1-6 (1983) and Scheidegger, A., et al., J. Antibiot. 37, 522-531 (1984) describe partial purification of the enzyme from C. acremonium while Jensen, S. E., et al., Antimicro. Agents Chemother. 24, 307-312, 1983, and Jensen, S. E., et al., J. Antibiot. 38, 263-265 (1985) describe the partial purification of the enzyme from S. clavuligerus. These studies involving the use of partially purified enzyme preparations have suggested that the ring expansion (DAOC formation) and the hydroxylation reaction (DAC formation) are catalyzed by two separate enzymes in prokaryotic S. clavuligerus and by a bifunctional expandase/hydroxylase in eukaryotic C. acremonium.
The purified enzyme provided by this invention demonstrates both expandase and hydroxylase functions and, accordingly, appears to be a bifunctional enzyme.